A. Partition chromatography

B. Ion-exchange chromatography

C. Affinity chromatography

D. Adsorption chromatography

Oligo(dT)-cellulose columns specifically bind poly(A) tails of mRNA through base-pairing with thymidine residues, making this a classic affinity chromatography application.

Question Analysis

mRNA isolation exploits the universal poly(A) tail (50-250 adenines) added post-transcriptionally to eukaryotic mRNAs. Oligo(dT) = stretch of ~12-25 thymidines covalently linked to cellulose, creating ligand-specific binding matrix.

Option Analysis

A. Partition chromatography

Incorrect. Partition separates by solubility between mobile and stationary liquid phases (e.g., paper chromatography). No specific biorecognition here—just physical partitioning.

B. Ion-exchange chromatography

Wrong. Ion-exchange separates by charge. Poly(A)-oligo(dT) binding is hydrogen bonding/base pairing, not electrostatic. High salt (0.5M NaCl) promotes binding by shielding charges.

C. Affinity chromatography

Correct. Affinity uses biological specificity: ligand (oligo dT) captures target (polyA mRNA) via reversible non-covalent interactions. Elution by low salt/heat disrupts A-T bonds.

D. Adsorption chromatography

No. Adsorption relies on surface attraction (polarity, charge). Here, binding is sequence-specific hybridization, not general surface adsorption.

Correct Answer

C. Affinity chromatography—oligo(dT)-cellulose exemplifies affinity purification: ligand specifically recognizes poly(A) tail.

Protocol Summary

1. Load: Total RNA in high-salt buffer → mRNA poly(A) hybridizes to oligo(dT)

2. Wash: Remove rRNA/tRNA (no polyA)

3. Elute: Low salt + 65°C → disrupt A-T base pairs, recover pure mRNA

GATE Prep Essential

Affinity chromatography definition: Stationary phase contains ligand with high, specific affinity for target molecule. Classic examples: Ni-NTA (His-tag), Protein A (antibodies), streptavidin-biotin, oligo(dT)-polyA.

Pro Tip: Remembers as “biological fishing”—bait (ligoand) catches specific prey (target). Regeneration: 0.1M NaOH strips column for reuse. Yields 1-5% of total RNA as mRNA.